Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that modulate excitability in several brain regions involved in the pathogenesis of epilepsy, including the hippocampus, neocortex, and thalamus. Accumulated evidence shows that downregulation of Ih, the current generated by HCN channels, causes neuronal hyperexcitability, and that genetic deletion of HCN1 channels, the main cortical and hippocampal subtype, accelerates the rate of epileptogenesis in acquired epilepsy models. More recent evidence shows that mutations in HCN1 underlie early life epileptic encephalopathy in some children with severe epilepsy and developmental delay. Thus HCN1 channelopathy occurs in both human genetic epilepsy and animal models of acquired epilepsy. In recent work from our laboratory, epilepsy induced by chemoconvulsant-induced status epilepticus (SE) was associated with loss of HCN1 channel expression that began within 1 hour post-SE, and persisted into chronic epilepsy. HCN1 channels were acutely internalized from the surface membrane of hippocampal pyramidal dendrites within the first hour following SE, well before the onset several days later of transcriptional downregulation of HCN1 mRNA expression. We now show for the first time that HCN1 channel surface expression is governed in a bidirectional fashion by protein kinase C (PKC) activity, with increased phosphorylation of HCN1 by PKC leading to decreased HCN1 surface expression. New preliminary data using mass spectrometry techniques show that HCN1 channels are minimally phosphorylated at rest, but undergo increased phosphorylation at S867 and S868 residues, which are also phosphorylated in naive tissue under conditions of PKC activation. These data suggest that the earliest manifestation of HCN1 channels dysfunction during epileptogenesis is PKC-mediated loss of surface expression and concomitant loss of HCN1-mediated current (Ih). Our goal in this R01 renewal application is to further understand the mechanisms of HCN1 channel dysfunction during the development of epilepsy. We propose to further study the phosphorylation signaling mechanisms underlying acute changes in HCN1 membrane trafficking after SE; determine whether these early events contribute to or initiate chronic HCN1 channelopathy; and characterize the effects of human HCN1 mutations on HCN1 channel biophysical properties. To do so, we will use state-of-the-art techniques including: mass spectrometry; viral transduction of mutant HCN1 channel expression in vivo; cellular electrophysiology in the dendrites of CA1 hippocampal pyramidal neurons; and analysis of a novel dataset of HCN1 mutations in a cohort of 800 children affected with epileptic encephalopathy of unknown cause.